Foxp3 fix perm kit

Manufactured by BD

Sourced in United States

The Foxp3 Fix/Perm kit is a laboratory equipment product designed for the intracellular staining and detection of the Foxp3 transcription factor. The kit provides the necessary reagents and protocols for the fixation, permeabilization, and staining of Foxp3 in cells.

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Lab products found in correlation

Lsrii machine, by BD (3 mentions) Fixable nir live dead dye, by Thermo Fisher Scientific (3 mentions) Cd3 pacific orange, by BD (2 mentions) Lsr 2, by BD (2 mentions) Cd4 qdot605, by BD (1 mentions) Il 2 apc, by BD (1 mentions) Cd4 qdot 605, by Thermo Fisher Scientific (1 mentions) Ifnγ alexa fluor 700, by BD (1 mentions) Tnf α pe cy7, by BD (1 mentions) Fcr block, by BD (1 mentions) Lsrfortessa, by BD (1 mentions) Anti cd16 32, by BioXCell (1 mentions) Cd11b af594 clone m1 70, by BioLegend (1 mentions) Bovine serum albumin (bsa), by BioXCell (1 mentions) Sp8 confocal microscope, by Leica (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Pimonidazole hydrochloride, by Hypoxyprobe (1 mentions)

6 protocols using foxp3 fix perm kit

Multiparametric Flow Cytometry of PBMC

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PBMC at 1×10⁶ cells/100μl were stimulated with peptide pools (F+G+H=NS3/4, I+L+M=NS5A/B) or PMA (phorbol 12-myristate 13-acetate)/ionomycin (50 and 500ng/ml respectively), or unstimulated (DMSO; 5ng/ml). Brefeldin-A was added (10μg/ml) 2-4 hours later, cells incubated overnight (37°C), stained with fixable-NIR live/dead dye (Life Technologies), fixed and permeabilised (Foxp3 Fix/Perm kit, BD Biosciences) then with CD3-Pacific Orange (1:50), CD4-Qdot605 (1:200), CD8-peridinin chlorophyll protein Cy5.5 (1:200), IFNγ-AlexaFluor700 (1:50), IL2-APC (allophycocyanin) (1:25), TNFα-PE (phycoerythrin)-Cy7 (1:25), IL17-PE (1:100) (Becton Dickson Sciences and Invitrogen) in perm-buffer (30 minutes). Flow cytometry was performed on a BD LSRII machine and analysed using FlowJo v.9.5.2 (Treestar).

Kelly C., Swadling L., Capone S., Brown A., Richardson R., Halliday J., von Delft A., Oo Y., Mutimer D., Kurioka A., Hartnell F., Collier J., Ammendola V., Sorbo M.D., Grazioli F., Esposito M.L., Marco S.D., Siani L., Traboni C., Hill A.V., Colloca S., Nicosia A., Cortese R., Folgori A., Klenerman P, & Barnes E. (2016). Chronic hepatitis C viral infection subverts vaccine‐induced T‐cell immunity in humans. Hepatology (Baltimore, Md.), 63(5), 1455-1470.

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Multiparametric Flow Cytometry of Stimulated PBMCs

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PBMCs at 1 × 10⁶ cells/100 μL were stimulated with peptide pools (F + G + H = NS3/4, I + L + M = NS5A/B) or phorbol 12‐myristate 13‐acetate/ionomycin (50 and 500 ng/mL, respectively) or unstimulated (DMSO, 5 ng/mL). Brefeldin‐A was added (10 μg/mL) 2‐4 hours later, and cells were incubated overnight (37°C), stained with fixable‐near infrared live/dead dye (Life Technologies), fixed, and permeabilized (Foxp3 Fix/Perm kit; BD Biosciences) with CD3‐Pacific Orange (1:50), CD4‐Qdot605 (1:200), CD8‐peridinin chlorophyll protein Cy5.5 (1:200), IFN‐γ‐AlexaFluor 700 (1:50), interleukin‐2 (IL‐2)‐allophycocyanin (1:25), tumor necrosis factor‐α (TNF‐α)‐phycoerythrin‐Cy7 (1:25), and IL‐17‐phycoerythrin (1:100; Becton Dickson Sciences and Invitrogen) in perm buffer (30 minutes). Flow cytometry was performed on a BD LSRII machine and analyzed using FlowJo v.9.5.2 (Treestar).

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Multiparametric T Cell Cytokine Profiling

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PBMC at 1 × 10⁶ cells/100 µL were stimulated with peptide pools (F + G + H = NS3/4, I + L + M = NS5A/B) or PMA (phorbol 12-myristate 13-acetate)/ionomycin (50 and 500 ng/mL respectively), or unstimulated (DMSO; 5 ng/mL). Brefeldin-A was added (10 µg/mL) 1 h later, cells incubated overnight (37 °C), stained with fixable-NIR live/dead dye (Life Technologies, Carlsbad, CA, USA), fixed and permeabilised (Foxp3 Fix/Perm kit, BD Biosciences, San Jose, CA, USA), then stained with CD3-Pacific Orange, CD4-Qdot605, CD8-peridinin chlorophyll protein (perCp) Cy5.5, IFNγ-AlexaFluor700 (1:50), IL2-APC (allophycocyanin), TNFα-PE (phycoerythrin)-Cy7, IL17-PE in perm-buffer (30 min room temperature (RT)). All flow cytometry was performed on a BD LSRII machine and analysed using FlowJo v9.5.2 (Treestar, Ashland, OR, USA).

Swadling L., Halliday J., Kelly C., Brown A., Capone S., Ansari M.A., Bonsall D., Richardson R., Hartnell F., Collier J., Ammendola V., Del Sorbo M., Von Delft A., Traboni C., Hill A.V., Colloca S., Nicosia A., Cortese R., Klenerman P., Folgori A, & Barnes E. (2016). Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection. Vaccines, 4(3), 27.

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Polyfunctionality Assessment of HCV-Specific T Cells

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PBMC at 1 × 10⁶ cells in 100 µl R10 were stimulated with peptide pools (F+G+H = NS3/4, I+L+M = NS5A/B) or PMA (phorbol 12-myristate 13-acetate)/ionomycin (50 and 500 ng/ml respectively), or unstimulated (DMSO). Brefeldin-A was added (10 µg/ml) 1 h later, cells were incubated overnight (37 °C), stained with fixable-NIR live/dead dye (Life Technologies), fixed (1% paraformaldehyde) and permeabilised (Foxp3 Fix/Perm kit, BD Biosciences) then stained with the following antibodies at room temperature for 30 mins: CD3-PO (Pacific Orange; Invitrogen, UCHT1), CD4-Qdot 605 (Invitrogen, S3.5) CD8-PB (Pacific Blue; BD biosciences, RPA-T8), IFNγ-Alexa Fluor 700 (BD biosciences, XMG1.2), IL-2-APC (BD biosciences, 5344.111), and TNFα-PE-Cy7 (BD biosciences, MAb11). Flow cytometry was performed using a BD LSRII and analysis by FlowJo (Tree Star) version 10.4.1. All ICS data are corrected for background (cytokine production in paired DMSO wells).
Pestle version 1.8 and Spice version 6.0 were used for background subtraction, data formatting, and data visualization for polyfunctionality assessment of ICS data. Samples with a total T cell response to HCV NS of <0.025% of CD4⁺ or CD8⁺ T cells were excluded. Pie base, means.

Capone S., Brown A., Hartnell F., Sorbo M.D., Traboni C., Vassilev V., Colloca S., Nicosia A., Cortese R., Folgori A., Klenerman P., Barnes E, & Swadling L. (2020). Optimising T cell (re)boosting strategies for adenoviral and modified vaccinia Ankara vaccine regimens in humans. NPJ Vaccines, 5, 94.

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Multiparametric Flow Cytometry Profiling

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Cells were incubated at 4°C with fixable live/dead aqua dye (Life Technology) for 20 minutes and then washed with PBS + 2% FBS. Cells were then treated with FcR block (BD Biosciences) for 10 minutes at RT prior to a 20-minute incubation with primary antibodies at 4°C. Samples used for intracellular staining were fixed and permeabilized with the FoxP3 fix/perm kit (BD Biosciences) prior to incubation at RT for 30 minutes with antibodies targeting intracellular proteins. Flow analyses were performed using either BD LSR II or BD LSRFortessa flow cytometers housed within the Unified Flow Cytometry Core at the University of Pittsburgh. Flow cytometry data were acquired using BD FACSDiva software and analyzed using FlowJo V.10. The antibodies used in flow-based studies are listed in Supplementary Table 1 with the gating strategies depicted in Supplementary Figure 1.

Filderman J.N., Taylor J.L., Wang J., Zhang Y., Singh P., Ross M.A., Watkins S.C., Nedal Al Bzour A., Karapetyan L., Kalinski P, & Storkus W.J. (2024). Antagonism of regulatory ISGs enhances the anti-melanoma efficacy of STING agonists. Frontiers in Immunology, 15, 1334769.

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Hypoxia Mapping in Murine Tissues

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Mice were injected with pimonidazole hydrochloride in PBS (80mg/kg; Hypoxyprobe) intraperitoneally 1.5 hours prior to sacrifice. Tissues were dissected and processed as described above. For flow cytometry studies, pimonidazole was visualized using anti-pimonidazole antibodies (Pacific Blue Mab-1 clone 4.3.11.3; Hypoxyprobe) after cells were fixed for 20 min at 4°C using the FOXP3 Fix/Perm kit (BD Biosciences), and washed in permeabilization buffer. For imaging studies, dissected tumors were embedded in OCT and sectioned into 10μm cryosections. Cryosections were stored at −80°C until further use. For immunostaining, sections were fixed in 4% PFA (Electron Microscopy Sciences) for 20 minutes at RT, followed by a rinse in PBS containing 1% BSA (Sigma). Sections were blocked in 1% BSA in PBS containing anti-CD16/32 (BioXCell) for 1 hour at RT, and washed. Sections were stained with CD11b-AF594 (clone M1/70;BioLegend), CD31-AF647 (clone 390;BioLegend) and Pacific Blue Mab-1 (clone 4.3.11.3;Hypoxyprobe) for 1 hour at RT, followed by a wash and mounted using Vectashield (Vector Laboratories) and sealed with nail polish. Images were acquired on a Leica SP8 confocal microscope. Data analysis was performed using Imaris (Bitplane).

Kersten K., Hu K.H., Combes A.J., Samad B., Harwin T., Ray A., Rao A.A., Cai E., Marchuk K., Artichoker J., Courau T., Shi Q., Belk J., Satpathy A.T, & Krummel M.F. (2022). Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer. Cancer cell, 40(6), 624-638.e9.

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